scholarly journals Epigenetic Small Molecules Rescue Nucleocytoplasmic Transport and DNA Damage Phenotypes in C9ORF72 ALS/FTD

2021 ◽  
Vol 11 (11) ◽  
pp. 1543
Author(s):  
Melina Ramic ◽  
Nadja S. Andrade ◽  
Matthew J. Rybin ◽  
Rustam Esanov ◽  
Claes Wahlestedt ◽  
...  
Keyword(s):  
Dna Damage ◽  
Small Molecules ◽  
Nuclear Import ◽  
Nuclear Export ◽  
Hdac Inhibitors ◽  
Nucleocytoplasmic Transport ◽  
Therapeutic Effects ◽  
Nuclear Localization Signals ◽  
Compound Libraries ◽  
Expansion Mutation

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease with available treatments only marginally slowing progression or improving survival. A hexanucleotide repeat expansion mutation in the C9ORF72 gene is the most commonly known genetic cause of both sporadic and familial cases of ALS and frontotemporal dementia (FTD). The C9ORF72 expansion mutation produces five dipeptide repeat proteins (DPRs), and while the mechanistic determinants of DPR-mediated neurotoxicity remain incompletely understood, evidence suggests that disruption of nucleocytoplasmic transport and increased DNA damage contributes to pathology. Therefore, characterizing these disturbances and determining the relative contribution of different DPRs is needed to facilitate the development of novel therapeutics for C9ALS/FTD. To this end, we generated a series of nucleocytoplasmic transport “biosensors”, composed of the green fluorescent protein (GFP), fused to different classes of nuclear localization signals (NLSs) and nuclear export signals (NESs). Using these biosensors in conjunction with automated microscopy, we investigated the role of the three most neurotoxic DPRs (PR, GR, and GA) on seven nuclear import and two export pathways. In addition to other DPRs, we found that PR had pronounced inhibitory effects on the classical nuclear export pathway and several nuclear import pathways. To identify compounds capable of counteracting the effects of PR on nucleocytoplasmic transport, we developed a nucleocytoplasmic transport assay and screened several commercially available compound libraries, totaling 2714 compounds. In addition to restoring nucleocytoplasmic transport efficiencies, hits from the screen also counteract the cytotoxic effects of PR. Selected hits were subsequently tested for their ability to rescue another C9ALS/FTD phenotype—persistent DNA double strand breakage. Overall, we found that DPRs disrupt multiple nucleocytoplasmic transport pathways and we identified small molecules that counteract these effects—resulting in increased viability of PR-expressing cells and decreased DNA damage markers in patient-derived motor neurons. Several HDAC inhibitors were validated as hits, supporting previous studies that show that HDAC inhibitors confer therapeutic effects in neurodegenerative models.

scholarly journals An Atp-Dependent, Ran-Independent Mechanism for Nuclear Import of the U1a and U2b′′ Spliceosome Proteins

2000 ◽  
Vol 148 (2) ◽  
pp. 293-304 ◽  
Author(s):  
Martin Hetzer ◽  
Iain W. Mattaj
Keyword(s):  
Adenosine Triphosphate ◽  
Nuclear Import ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Fraction ◽  
Gtp Hydrolysis ◽  
Permeabilized Cells ◽  
Nuclear Localization Signals ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Ribonucleoprotein

Nuclear import of the two uracil-rich small nuclear ribonucleoprotein (U snRNP) components U1A and U2B′′ is mediated by unusually long and complex nuclear localization signals (NLSs). Here we investigate nuclear import of U1A and U2B′′ in vitro and demonstrate that it occurs by an active, saturable process. Several lines of evidence suggest that import of the two proteins occurs by an import mechanism different to those characterized previously. No cross competition is seen with a variety of previously studied NLSs. In contrast to import mediated by members of the importin-β family of nucleocytoplasmic transport receptors, U1A/U2B′′ import is not inhibited by either nonhydrolyzable guanosine triphosphate (GTP) analogues or by a mutant of the GTPase Ran that is incapable of GTP hydrolysis. Adenosine triphosphate is capable of supporting U1A and U2B′′ import, whereas neither nonhydrolyzable adenosine triphosphate analogues nor GTP can do so. U1A and U2B′′ import in vitro does not require the addition of soluble cytosolic proteins, but a factor or factors required for U1A and U2B′′ import remains tightly associated with the nuclear fraction of conventionally permeabilized cells. This activity can be solubilized in the presence of elevated MgCl2. These data suggest that U1A and U2B′′ import into the nucleus occurs by a hitherto uncharacterized mechanism.

scholarly journals The Ebola Virus Interferon Antagonist VP24 Undergoes Active Nucleocytoplasmic Trafficking

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1650
Author(s):  
Angela R. Harrison ◽  
Cassandra T. David ◽  
Stephen M. Rawlinson ◽  
Gregory W. Moseley
Keyword(s):  
Nuclear Import ◽  
Nuclear Export ◽  
Ebola Virus ◽  
Molecular Mapping ◽  
Nucleocytoplasmic Transport ◽  
Cytoplasmic Localization ◽  
Nucleocytoplasmic Trafficking ◽  
Nuclear Trafficking ◽  
C Terminus ◽  
Import Receptors

Viral interferon (IFN) antagonist proteins mediate evasion of IFN-mediated innate immunity and are often multifunctional, with distinct roles in viral replication. The Ebola virus IFN antagonist VP24 mediates nucleocapsid assembly, and inhibits IFN-activated signaling by preventing nuclear import of STAT1 via competitive binding to nuclear import receptors (karyopherins). Proteins of many viruses, including viruses with cytoplasmic replication cycles, interact with nuclear trafficking machinery to undergo nucleocytoplasmic transport, with key roles in pathogenesis; however, despite established karyopherin interaction, potential nuclear trafficking of VP24 has not been investigated. We find that inhibition of nuclear export pathways or overexpression of VP24-binding karyopherin results in nuclear localization of VP24. Molecular mapping indicates that cytoplasmic localization of VP24 depends on a CRM1-dependent nuclear export sequence at the VP24 C-terminus. Nuclear export is not required for STAT1 antagonism, consistent with competitive karyopherin binding being the principal antagonistic mechanism, while export mediates return of nuclear VP24 to the cytoplasm where replication/nucleocapsid assembly occurs.

Signal-mediated nuclear transport in the amoeba

1999 ◽  
Vol 112 (12) ◽  
pp. 2043-2048 ◽  
Author(s):  
C.M. Feldherr ◽  
D. Akin
Keyword(s):  
Nuclear Import ◽  
Nuclear Export ◽  
Transport Process ◽  
Nuclear Transport ◽  
Amoeba Proteus ◽  
Regulatory Requirements ◽  
Nuclear Localization Signals ◽  
Evolutionary Changes ◽  
Composition And Structure ◽  
Cytosolic Factors

The evolutionary changes that occur in signal-mediated nuclear transport would be expected to reflect an increasing need to regulate nucleocytoplasmic exchanges as the complexity of organisms increases. This could involve changes in both the composition and structure of the pore complex, as well as the cytosolic factors that mediate transport. In this regard, we investigated the transport process in amoebae (Amoeba proteus and Chaos carolinensis), primitive cells that would be expected to have less stringent regulatory requirements than more complex organisms. Colloidal gold particles, coated with bovine serum albumin (BSA) conjugated with simple (large T) nuclear localization signals (NLSs), bipartite (nucleoplasmin) NLSs or mutant NLSs, were used to assay nuclear import. It was found that in amoebae (1) the diameter of the particles that are able to enter the nucleoplasm is significantly less than in vertebrate cells, (2) the simple NLS is more effective in mediating nuclear import than the bipartite NLS, and (3) the nucleoporins do not appear to be glycosylated. Evidence was also obtained suggesting that, in amoebae, the simple NLS can mediate nuclear export.

scholarly journals Identification of Nuclear Import and Export Signals within Fli-1: Roles of the Nuclear Import Signals in Fli-1-Dependent Activation of Megakaryocyte-Specific Promoters

2005 ◽  
Vol 25 (8) ◽  
pp. 3087-3108 ◽  
Author(s):  
Wei Hu ◽  
Alana S. Philips ◽  
Juliana C. Kwok ◽  
Michael Eisbacher ◽  
Beng H. Chong
Keyword(s):  
Nuclear Import ◽  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Nuclear Accumulation ◽  
Leptomycin B ◽  
Nuclear Localization Signals ◽  
Importin Alpha ◽  
Friend Leukemia ◽  
Important Regulator ◽  
Ets Domain

ABSTRACT The Ets factor Friend leukemia integration 1 (Fli-1) is an important regulator of megakaryocytic (Mk) differentiation. Here, we demonstrate two novel nuclear localization signals (NLSs) within Fli-1: one (NLS1) is located at the N terminus, and another (NLS2) is within the Ets domain. Nuclear accumulation of Fli-1 reflected the combined functional effects of the two discrete NLSs. Each NLS can independently direct nuclear transport of a carrier protein, with mutations within the NLSs affecting nuclear accumulation. NLS1 has a bipartite motif, whereas the NLS2 region contains a nonclassical NLS. Both NLSs bind importin alpha (IMPα) and IMPβ, with NLS1 and NLS2 being predominantly recognized by IMPα and IMPβ, respectively. Fli-1 also contains one nuclear export signal. Leptomycin B abolished its cytoplasmic accumulation, showing CRM1 dependency. We demonstrate that Ets domain binding to specific target DNA effectively blocks IMP binding, indicating that the targeted DNA binding plays a role in localizing Fli-1 to its destination and releasing IMPs for recycling back to the cytoplasm. Finally, by analyzing full-length Fli-1 carrying NLS1, NLS2, and combined NLS1-NLS2 mutations, we conclude that two functional NLSs exist in Fli-1 and that each NLS is sufficient to target Fli-1 to the nucleus for activation of Mk-specific genes.

scholarly journals The Ran GTPase Gradient Protects the Nucleolus from Aging-Associated Morphological Changes

2020 ◽  
Author(s):  
Bartlomiej Remlein ◽  
Bryce M. Paschal
Keyword(s):  
Molecular Mass ◽  
Nuclear Import ◽  
Nuclear Export ◽  
Cultured Cells ◽  
Nucleocytoplasmic Transport ◽  
High Molecular Mass ◽  
Regulatory Function ◽  
Chemical Induction ◽  
Ran Gtpase ◽  
Passage Number

ABSTRACTIn the context of its regulatory function for nucleocytoplasmic transport, the Ran GTPase undergoes cycles of nuclear import, GTP loading, nuclear export, and GTP hydrolysis. These reactions give rise to a nuclear:cytoplasmic (N:C) Ran gradient. In Hutchinson-Gilford Progeria Syndrome, disruption of the Ran gradient suppresses nuclear import of high molecular mass complexes by reducing the nuclear concentration of Ran. Here, we report that cells undergoing senescence, as a consequence of passage number, chemical induction, and altered nuclear lamina structure, all display a Ran gradient disruption quantitatively similar to that observed in Progeria patient cells. We found that the Ran gradient is critical for maintenance of nucleolar structure, as its disruption increases the size and decreases the average number of nucleoli per cell. Nucleolar number and size are biomarkers of longevity in diverse organisms, thus the nuclear level of Ran may be important for the nucleolar morphology in aging. The contribution of the Ran gradient includes regulating import of nucleolin and nucleophosmin, nucleolar proteins that assemble into high molecular mass complexes. The steepness of the Ran gradient is highly dependent on nuclear heterochromatin, which is reduced by passage number and chemical induction of senescence in cultured cells, and is known to decline during normal aging. Our data suggest that the Ran gradient senses nuclear heterochromatin, and through its function as a transport regulator, helps maintain the protein composition and structure of the nucleolus.

A functional study of nucleocytoplasmic transport signals of the EhNCABP166 protein from Entamoeba histolytica

Parasitology ◽  
2012 ◽  
Vol 139 (13) ◽  
pp. 1697-1710 ◽  
Author(s):  
R. URIBE ◽  
J. ALMARAZ BARRERA MA DE ◽  
M. ROBLES-FLORES ◽  
G. MENDOZA HERNÁNDEZ ◽  
A. GONZÁLEZ-ROBLES ◽  
...  
Keyword(s):  
Entamoeba Histolytica ◽  
Nuclear Localization ◽  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Cytoplasmic Domain ◽  
Nucleocytoplasmic Transport ◽  
Actin Binding ◽  
Functional Study ◽  
Nuclear Localization Signals ◽  
Export Signal

SUMMARYEhNCABP166 is an Entamoeba histolytica actin-binding protein that localizes to the nucleus and cytoplasm. Bioinformatic analysis of the EhNCABP166 amino acid sequence shows the presence of 3 bipartite nuclear localization signals (NLS) and a nuclear export signal (NES). The present study aimed to investigate the functionality of these signals in 3 ways. First, we fused each potential NLS to a cytoplasmic domain of ehFLN to determine whether the localization of this domain could be altered by the presence of the NLSs. Furthermore, the localization of each domain of EhNCABP166 was determined. Similarly, we generated mutations in the first block of bipartite signals from the domains that contained these signals. Additionally, we added an NES to 2 constructs that were then evaluated. We confirmed the intranuclear localization of EhNCABP166 using transmission electron microscopy. Fusion of each NLS resulted in shuttling of the cytoplasmic domain to the nucleus. With the exception of 2 domains, all of the evaluated domains localized within the nucleus. A mutation in the first block of bipartite signals affected the localization of the domains containing an NLS. The addition of an NES shifted the localization of these domains to the cytoplasm. The results presented here establish EhNCABP166 as a protein containing functional nuclear localization signals and a nuclear export signal.

scholarly journals Karyopherin α-2 Mediates MDC1 Nuclear Import through a Functional Nuclear Localization Signal in the tBRCT Domain of MDC1

2020 ◽  
Vol 21 (7) ◽  
pp. 2650
Author(s):  
Kamalakannan Radhakrishnan ◽  
Seon-Joo Park ◽  
Seok Won Kim ◽  
Gurusamy Hariharasudhan ◽  
Seo-Yeon Jeong ◽  
...  
Keyword(s):  
Dna Damage ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Ring Finger ◽  
Nucleocytoplasmic Transport ◽  
Ring Finger Protein ◽  
Finger Protein ◽  
Localization Signal ◽  
Functional Nuclear Localization Signal

Mediator of DNA damage checkpoint protein 1 (MDC1) plays a vital role in DNA damage response (DDR) by coordinating the repair of double strand breaks (DSBs). Here, we identified a novel interaction between MDC1 and karyopherin α-2 (KPNA2), a nucleocytoplasmic transport adaptor, and showed that KPNA2 is necessary for MDC1 nuclear import. Thereafter, we identified a functional nuclear localization signal (NLS) between amino acid residues 1989–1994 of the two Breast Cancer 1 (BRCA1) carboxyl-terminal (tBRCT) domain of MDC1 and demonstrated disruption of this NLS impaired interaction between MDC1 and KPNA2 and reduced nuclear localization of MDC1. In KPNA2-depleted cells, the recruitment of MDC1, along with the downstream signaling p roteins Ring Finger Protein 8 (RNF8), 53BP1-binding protein 1 (53BP1), BRCA1, and Ring Finger Protein 168 (RNF168), to DNA damage sites was abolished. Additionally, KPNA2-depleted cells had a decreased rate of homologous recombination (HR) repair. Our data suggest that KPNA2-mediated MDC1 nuclear import is important for DDR signaling and DSB repair.

scholarly journals Involvement of Nuclear Import and Export Factors in U8 Box C/D snoRNP Biogenesis

2007 ◽  
Vol 27 (20) ◽  
pp. 7018-7027 ◽  
Author(s):  
Nicholas J. Watkins ◽  
Ira Lemm ◽  
Reinhard Lührmann
Keyword(s):  
Rna Interference ◽  
Nuclear Import ◽  
Nuclear Export ◽  
Ribosome Biogenesis ◽  
Nucleocytoplasmic Transport ◽  
Differential Association ◽  
Import And Export ◽  
Snornp Biogenesis ◽  
Cellular Compartments ◽  
Distinct Distribution

ABSTRACT Box C/D snoRNPs, factors essential for ribosome biogenesis, are proposed to be assembled in the nucleoplasm before localizing to the nucleolus. However, recent work demonstrated the involvement of nuclear export factors in this process, suggesting that export may take place. Here we show that there are distinct distributions of U8 pre-snoRNAs and pre-snoRNP complexes in HeLa cell nuclear and cytoplasmic extracts. We observed differential association of nuclear export (PHAX, CRM1, and Ran) factors with complexes in the two extracts, consistent with nucleocytoplasmic transport. Furthermore, we show that the U8 pre-snoRNA in one of the cytoplasmic complexes contains an m3G cap and is associated with the nuclear import factor Snurportin1. Using RNA interference, we show that loss of either PHAX or Snurportin1 results in the incorrect localization of the U8 snoRNA. Our data therefore show that nuclear export and import factors are directly involved in U8 box C/D snoRNP biogenesis. The distinct distribution of U8 pre-snoRNP complexes between the two cellular compartments together with the association of both nuclear import and export factors with the precursor complex suggests that the mammalian U8 snoRNP is exported during biogenesis.

scholarly journals The Ebola virus interferon antagonist VP24 undergoes active nucleocytoplasmic trafficking

2020 ◽  
Author(s):  
Angela R. Harrison ◽  
Gregory W. Moseley
Keyword(s):  
Viral Replication ◽  
Immune Evasion ◽  
Nuclear Import ◽  
Nuclear Export ◽  
Ebola Virus ◽  
Molecular Mapping ◽  
Case Fatality ◽  
Nucleocytoplasmic Transport ◽  
Nucleocytoplasmic Trafficking ◽  
Nuclear Trafficking

AbstractViral interferon (IFN) antagonist proteins mediate evasion of IFN-mediated innate immunity and are often multifunctional, having distinct roles in viral replication processes. Functions of the Ebola virus (EBOV) IFN antagonist VP24 include nucleocapsid assembly during cytoplasmic replication and inhibition of IFN-activated signalling by STAT1. For the latter, VP24 prevents STAT1 nuclear import via competitive binding to nuclear import receptors (karyopherins). Many viral proteins, including proteins from viruses with cytoplasmic replication cycles, interact with the trafficking machinery to undergo nucleocytoplasmic transport, with key roles in pathogenesis. Despite established karyopherin interaction, the nuclear trafficking profile of VP24 has not been investigated. We find that VP24 becomes strongly nuclear following overexpression of karyopherin or inhibition of nuclear export pathways. Molecular mapping indicates that cytoplasmic localisation of VP24 depends on a CRM1-dependent nuclear export sequence at the VP24 C-terminus. Nuclear export is not required for STAT1 antagonism, consistent with competitive karyopherin binding being the principal antagonistic mechanism while export mediates return of nuclear VP24 to the cytoplasm for replication functions. Thus, nuclear export of VP24 might provide novel targets for antiviral approaches.ImportanceEbola virus (EBOV) is the causative agent of ongoing outbreaks of severe haemorrhagic fever with case-fatality rates between 40 and 60%. Proteins of many viruses with cytoplasmic replication cycles similar to EBOV interact with the nuclear trafficking machinery, resulting in active nucleocytoplasmic shuttling important to immune evasion and other intranuclear functions. However, exploitation of host trafficking machinery for nucleocytoplasmic transport by EBOV has not been directly examined. We find that the EBOV protein VP24 is actively trafficked between the nucleus and cytoplasm, and identify the specific pathways and sequences involved. The data indicate that nucleocytoplasmic trafficking is important for the multifunctional nature of VP24, which has critical roles in immune evasion and viral replication, identifying a new mechanism in infection by this highly lethal pathogen, and potential target for antivirals.

scholarly journals Determination of the Functional Domain Organization of the Importin α Nuclear Import Factor

1998 ◽  
Vol 143 (2) ◽  
pp. 309-318 ◽  
Author(s):  
Andrea Herold ◽  
Ray Truant ◽  
Heather Wiegand ◽  
Bryan R. Cullen
Keyword(s):  
Nuclear Import ◽  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Point Mutations ◽  
T Antigen ◽  
Functional Domain ◽  
Nuclear Localization Signals ◽  
Importin Α ◽  
Export Signal

Although importin α (Imp α) has been shown to act as the receptor for basic nuclear localization signals (NLSs) and to mediate their recruitment to the importin β nuclear import factor, little is known about the functional domains present in Imp α, with the exception that importin β binding is known to map close to the Imp α NH2 terminus. Here, we demonstrate that sequences essential for binding to the CAS nuclear export factor are located near the Imp α COOH terminus and include a critical acidic motif. Although point mutations introduced into this acidic motif inactivated both CAS binding and Imp α nuclear export, a putative leucine-rich nuclear export signal proved to be neither necessary nor sufficient for Imp α nuclear export. Analysis of sequences within Imp α that bind to the SV-40 T antigen NLS or to the similar LEF-1 NLS revealed that both NLSs interact with a subset of the eight degenerate armadillo (Arm) repeats that form the central part of Imp α. However, these two NLS-binding sites showed only minimal overlap, thus suggesting that the degeneracy of the Arm repeat region of Imp α may serve to facilitate binding to similar but nonidentical basic NLSs. Importantly, the SV-40 T NLS proved able to specifically inhibit the interaction of Imp α with CAS in vitro, thus explaining why the SV-40 T NLS is unable to also function as a nuclear export signal.

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